PROJECT SUMMARY While cancer has traditionally been viewed as a disease process based exclusively on genetic aberrations, increasing evidence has demonstrated that epigenetic alterations contribute to the pathogenesis and progression of many types of cancer. One emerging epigenetic driver of cancer is the superenhancer, which is defined as a cluster of typical enhancers in close genomic proximity. The significance of superenhancers is supported by their roles in determining cell state and identity through regulation of lineage-specific gene expression, with enrichment in disease-associated genetic variation. Superenhancers identified from cancer cells identify tumor- associated genes in a number of cancer types, including multiple brain tumors. Due to the close association between superenhancer structure and biological function, alterations in disease-specific superenhancers may reveal insights into the molecular mechanisms of disease pathogenesis. Glioblastoma is the most common and most aggressive primary brain tumor, with poor responses to all therapeutic modalities despite intensive research. One major contributor to the poor prognosis of glioblastoma is the presence of stem-like cancer cells, often called cancer stem cells. Cancer stem cells are functionally defined by their abilities to self-renew, differentiate, and form tumors upon transplantation. Cancer stem cells contribute to resistance to radiation and chemotherapy, as well as maintenance of tumor heterogeneity and angiogenesis. Thus, cancer stem cells have become an important target for the design of novel therapeutic strategies. To better understand key regulators of glioma stem cell identity, an original super-enhancer screen identified genes that are epigenetically upregulated in glioma stem cells and for which elevated expression is associated with poor patient prognosis. Leveraging chromatin landscape analysis of patient-derived glioblastoma stem cells, I identified putative superenhancers that are associated with poor patient prognosis. Targeting the genes associated with the superenhancers revealed a loss of viability, suggesting potential value in this discovery effort. This approach revealed a cohort of potential super-enhancer associated genes that I propose for further study. I will investigate the transcription factor network that regulates the expression of these genes including enhancer elements, transcription factor occupancy, and super-enhancer structure. The second aim will define the role of the superenhancer-associated genes in glioma cell survival, self-renewal capacity, and tumor formation in both in vitro and in vivo settings. The third aim will elucidate the molecular mechanism by which these genes promote tumorigenesis. These approaches will lead to a greater understanding of the epigenetic features that define the glioma stem cell state and inform the development of novel therapeutics